Name: quantification of endogenous auxin and cytokinin during internode culture of ipecac
Uploaded: 2018-03-15T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Nous sommes reconnaissants à M. Akira Murakami, du département de Biosciences appliquées, Université de Toyo et M. Koudai Taniguchi de l’Agricultural Technology Center de Gunma pour leur assistance technique. Nous sommes également reconnaissants au professeur Shosaku Kashiwada et Dr Uma Maheswari Rajagopalan, Université Toyo pour leurs suggestions. Cette étude a été financée en partie par le centre de recherche pour la vie et Sciences de l’environnement, l’Université Toyo.

Remarque : Ipéca (c. ipécacuanha) a été utilisée dans cette étude, car elle facilite l’analyse des phytohormones endogènes.
1. croissance Conditions d’induire des pousses adventives d’ipéca
<ol><li>Préparer un milieu B5 sans phytohormone ajusté à pH 5,710et ajoutez la gomme Gellane 0,2 %. Stériliser à l’autoclave.</li>
	<li>Verser 25 mL de milieu stérilisé dans une boîte de Petri stérile (90 × 20 mm).</li>
	<li>Couper des segments i...

<ol>
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Pour identifier la distribution des phytohormones impliqués dans l’organogenèse, c’est important d’utiliser des matières végétales dans lesquelles organogenèse peut être observé sur un milieu sans phytohormone, parce que quand les phytohormones sont appliqués exogène d’explants pour induire tiges ou racines, ils affectent l’explant entier, rendant difficile d’évaluer la plasticité inhérente des plantes dans la différenciation cellulaire et l’organogénèse. Des pousses adventives peuvent être...

Gottlieb Haberlandt (1854-1945) a proposé le concept de « totipotence », par quelles plantes cellules peuvent diviser, différencier et régénérer des plantes entières même après leur différenciation préalable en types spécifiques de cellules de plantes matures1. En culture de tissus, si la régénération des plantes peut être induite ou non dépend de la combinaison et la concentration des phytohormones exogènes appliquées dans le milieu de croissance. Skoog et Miller ont constaté que des pousses adventives peuvent être induites de tabac cals sur milieu de culture contenant un taux élevé de CKs d’auxines, tandis que des racines adventives pouvaient être induites sur un milieu contenant un faible ratio2. Depuis cette conclusion, culture de tissus a été largement utilisée pour la propagation des cultures d’importance économique et pour la régénération des plantes transgéniques3. Des pousses adventives peuvent être induites de tissus autres que le méristème apical, tels que des feuilles, des racines et des entre-noeuds. Phytohormone traitement est nécessaire à l’induction des pousses adventives dans la plupart des espèces végétales. Toutefois, les combinaisons et les concentrations optimales diffèrent par espèce et entre les tissus utilisés comme explants. Ainsi, beaucoup d’efforts va en déterminer les concentrations optimales et les combinaisons de phytohormones pour des expériences.
Carapichea ipecacuanha (Brot.) L. Andersson (ipéca) est une plante médicinale qui contient des alcaloïdes tels qu’émétine et Céphéline, surtout dans les racines4. Extraits de racines sont utilisées comme expectorant, émétique et un amoebicide5. Bien que l’ipéca pousse naturellement dans les forêts tropicales du Brésil, il est réticent à mettre les graines dans la culture, et le taux de germination diminue pendant le stockage de semences au Japon, avec son de climat plus froid6. Au lieu de cela, il est propagé par culture de tissus, dans laquelle adventives tirer formation sur entre-noeuds est la plus efficace méthode de7,,8. Fait intéressant, les pousses adventives peuvent être induites chez cette espèce sans phytohormone traitement8.
Des pousses adventives sont forment sur l’épiderme dans la région apicale des segments internodales sans obtention, mais pas dans la région basale9. Cette différence indique les polarités de tissus dans les segments internodales, qui est probablement en vertu du règlement phytohormonal. Le système de culture d’ipéca permet une occasion unique d’analyser les changements dans les niveaux endogènes phytohormone au cours de la formation de pousses adventives. Ici, nous présentons notre méthode pour l’analyse des niveaux endogènes d’une auxine (acide indole 3-acétique (AIA)) et quatre CKs (isopentényl adénine (iP), isopentényl adénine riboside (iPR), trans-zéatine (tZ) et trans-zéatine riboside (tZR)) dans les segments internodales grâce à l’utilisation de CL-SM/SM.

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		<col />
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H5]indole-3-acetic acid</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">031 1531</td>
			<td style="width:167px;">Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H5]trans-zeatin</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0301</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">[2H5]trans-zeatin riboside</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0311</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H6]N6-isopentenyl adenine</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0161</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">[2H6]N6-isopentenyl adenosine</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0171</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">indole-3-acetic acid</td>
			<td style="width:176px;">Wako</td>
			<td>098 00181</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trans-zeatin</td>
			<td style="width:176px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">Z0876 5MG</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trans-zeatin riboside</td>
			<td style="width:176px;">Wako</td>
			<td>262 01081</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N6-isopentenyl adenine</td>
			<td style="width:176px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">D7674 1G</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N6-isopentenyl adenosine</td>
			<td style="width:176px;">ACROS ORGANICS</td>
			<td style="width:119px;">22648 1000</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">acetonitrile hypergrade for LC-MS LiChrosolv</td>
			<td style="width:176px;">MERCK</td>
			<td style="width:119px;">1.00029.1000</td>
			<td>solvent for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Water for chromatography LiChrosolv</td>
			<td style="width:176px;">MERCK</td>
			<td style="width:119px;">1.15333.1000</td>
			<td>solvent for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC</td>
			<td style="width:176px;">SHIMADZU</td>
			<td style="width:119px;">Prominence</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS</td>
			<td style="width:176px;">Sciex</td>
			<td style="width:119px;">3200QTRAP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis HLB 30 mg/1 cc</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">WAT094225</td>
			<td>cartridge column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis MCX 30 mg/1 cc</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186000252</td>
			<td>cartridge column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">screw neck total recovery vial</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186002805</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">blue, 12 x 32mm screw neck cap and PTFE/silicone septum</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186000274</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Acquity UPLC BEH C18, 2.1x100 mm</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186002350</td>
			<td>UPLC column</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Proshell 120 EC-C18, 2.1x50 mm</td>
			<td style="width:176px;">Agilent</td>
			<td style="width:119px;">699775-902</td>
			<td>UPLC column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digital microscope</td>
			<td style="width:176px;">Leica</td>
			<td style="width:119px;">DHS1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TissueLyser II</td>
			<td style="width:176px;">QIAGEN</td>
			<td style="width:119px;">85300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:176px;">Feather</td>
			<td style="width:119px;">No. 22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel handle</td>
			<td style="width:176px;">Feather</td>
			<td style="width:119px;">No. 4</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Savant SpeedVac/Refregerated vapor trap</td>
			<td style="width:176px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">SPD111/RVT4104</td>
			<td>vacuum concentrartor</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Disposable glass tobe (13x100 mm)</td>
			<td style="width:176px;">IWAKI</td>
			<td style="width:119px;">9832-1310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile petri dish</td>
			<td style="width:176px;">INA OPTICA</td>
			<td style="width:119px;">I-90-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of endogenous auxin and cytokinin during internode culture of ipecac

La formation de pousses adventives est une technique importante pour la propagation des cultures d’importance économique et à la régénération de plantes transgéniques. Phytohormone traitement est nécessaire à l’induction des pousses adventives dans la plupart des espèces. Si des pousses adventives peuvent être induites sont déterminée par l’équilibre entre auxine et cytokinine (CK) niveaux. Beaucoup d’efforts va dans la détermination des concentrations optimales et des combinaisons de phytohormones dans chaque tissu utilisé comme explants et dans chacune des espèces végétales. Dans ipecac, toutefois, des pousses adventives peuvent être induites sur internodales segments dans le milieu de culture sans traitement phytohormone. Cela permet la plasticité inhérente d’ipéca pour la différenciation des cellules doit être évaluée. Pour induire des pousses adventives en ipecac, nous cultivées internodales segments à 24 ° C sous 15 µmol m−2 s−1 de lumière dans un cycle de sombre lumière/10 h de 14 h sur un milieu B5 sans phytohormone solidifié avec gomme Gellane 0,2 % pendant 5 semaines. Pour étudier la dynamique phytohormone au cours de la formation de pousses adventives, nous avons mesuré l’acide indole 3-acétique endogène et CKs dans les segments par spectrométrie de masse en tandem par chromatographie liquide LC-MS/MS. Cette méthode permet l’analyse de l’acide indole 3-acétique endogène et CKs niveaux de manière simple. Il peut être appliqué afin d’étudier la dynamique de l’auxine endogène et CK pendant l’organogenèse chez d’autres espèces végétales.

À la 1st semaine, aucun des pousses adventives n’avaient formé. À la 2ème semaine, petites pousses sont apparus. À la 3rd et 4ème semaines, le nombre de pousses accru pour la plupart dans les régions apicales (I et II) (Figure 2A). À la 5e semaine, le nombre de pousses était approximativement 7 en région I et 5 dans la région II (Figure 2...

Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Quantification de l’endogène auxine et cytokinine durant la Culture entre-nœud d’ipéca

Des pousses adventives peuvent être induites sur les segments internodales d’ipéca sans traitement phytohormone. Pour évaluer la dynamique phytohormone au cours de la formation de pousses adventives, nous avons mesuré endogène auxine et cytokinine dans des segments internodales par LC-MS/MS.

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. ...

The overall goal of this procedure is to describe how endogenous phytohormone levels change during adventitious shoot formation in ipecac. This method can help answer key questions related to tissue regeneration in the plant tissue culture wheel. The main advantage of this technique is the endogenous level of Indole-3-acetic acid and cytokinines can be simply analyzed with high sensitivity.The implication of this technique extend throughout establishment of plant tissue degeneration because changes in endogenous phytohormone level trigger adventitious shoot formation. To begin, prepare a phytohormone-free B5 medium and add 0.2 percent gellan gum. Then, autoclave the medium to sterilize it.Using a surgical scalpel on a sterile acrylic plate, slice eight millimeter internodal segments of ipecac plantlets and place them on the medium. Then, culture the ipecac segments at 24 degrees Celsius under 15 micromole per square meter seconds of light in a 14-hour light, 10-hour dark cycle for five weeks. After culturing the ipecac segments, identify regions one through four in each segment.Once a week, use a microscope to count the number of adventitious shoots in each region. First, place a five millimeter zirconia bead into four two-milliliter sample tubes. Then, use a surgical scalpel on a sterile acrylic plate to cut the ipecac segments into regions I through IV.Then collect eight segments from each region into one sample tube.Weigh each of the samples and freeze them in liquid nitrogen. Next, use a bead-based homogenizer to crush the frozen samples. Use a vortex mixer to suspend the samples in one milliliter of the acetonitrile with 500 picograms of each internal standard of oxen and CKs.Home the samples at four degrees Celsius for one hour. And then centrifuge the samples at 3500 G for five minutes at room temperature. Wash the pellet in 80 percent acetal nitrile with one percent acetic acid.Then repeat the centrifugation and use a disposable class tube to combine the supernatants. Add 600 microliters of water with one percent acetic acid to the combined supernatant. Then use a vacuum concentrator to evaporate the acetal nitrile.Load the sample solutions into individual equilibrated HLB cartridges. And wash the cartridges with one milliliter of water with one percent acetic acid. Then allude all of the hormones with two milliliters of 80 percent acetyl nitrile with one percent acetic acid in a glass tube.Using a vacuum concentrator, evaporate the acetal nitrile to obtain extract in water with one percent acetic acid. Load the sample solutions into individual equilibrated MCX cartridges and wash the cartridges with one milliliter of water with one percent acetic acid. Then allude IAA in a glass tube, with two milliliters of 30 percent acetal nitrile with one percent acetic acid.Then wash the cartridges with two milliliters of 80 percent acetal nitrile with one percent acetic acid. Next, wash the cartridges with two milliliters of water and one milliliter of water with five percent aqueous ammonia in a draft chamber. Allude the CKs in a glass tube with two milliliters of 60 percent acetal nitrile with five percent aqueous ammonia in a draft chamber.Then use a vacuum concentrator to evaporate the solvent of each hormone fraction. Stir the samples at minus thirty degrees Celsius until LCMSMS analysis. First, dissolve each hormone extract in a tube with 600 microliters of methanol.Then transfer the solution to a screw-neck total recovery vial and use a vacuum concentrator to evaporate the solvent. Dissolve the IAA fraction in 20 microliters of 30 percent acetal nitrile and the CK fractions in 20 microliters of water with one percent acetic acid. Next use a triple quadrupole MS system equipped with an HPLC system to analyze the samples in positive ion mode.Finally quantify endogenous IAA in CK levels against a standard curve of the ration of unlabeled to deuterium label standards. In this protocol endogenous oxidant cytokinin in adventitious chutes was measured via LCMSMS for five weeks. At the first week, no adventitious chutes had formed and the first small chutes appeared during the second week.During the third and fourth weeks, the number of chutes increased mostly in regents one and two. During the fifth week the number of chutes was approximately seven in region one, and five in region two. Before culture, the IAA level was slightly higher in region one than in regions two, three, and four.During the first week, the IAA level increased greatly in region four, and decreased slightly in regions one, two, and three. By five weeks of culture an IAA concentration gradient emerged. With levels increasing from region one to region four.Before culture, there were only trace levels of most of the CKs. At the first week, the tZR level increased to 13.8 picograms per milligram in region two. And 18.1 picograms per milligram in region three.In contrast the levels of tZ, iP and iPR changed only slightly during culture. While attempting this procedure it's important to remember that endogenous part hormone are stable. After it's developed man, this technique paves the way for researchers in the field of plant tissue culture.To explore adventitious formation in other organisms. After watching this video, you should have a good understanding of how to analyze endogenous Indole-3-acetic acid and cytokinines.

Research

JoVE Journal

Environment

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

Méthode EPA 1615. Mesure de entérovirus et Norovirus présence dans l&#39;eau par Culture et RT-qPCR. I. Prélèvement des échantillons de virus

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Recherche

Environnement

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Quantification des métaux lourds et d&#39;autres contaminants inorganiques sur la productivité des microalgues

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

ARN-ADN Extraction simultanée de sédiments côtiers et Quantification de 16S ARNr et Transcriptions par PCR en temps réel

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

Capturant pondérée en fonction du débit de l’eau et les particules en suspension de canaux agricole pendant les événements de Drainage

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

L’effet de l’Application d’huile essentielle de thym sur la charge microbienne pendant le séchage de la viande

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction

Distribution des contraintes lors de la Compression froide des roches et des agrégats de minéraux à l’aide de la Diffraction des rayons x axés sur le rayonnement Synchrotron

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) ...

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Isolement chimique, Quantification et séparation des lipides de la peau des Reptiles

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Split Point Analysis and Uncertainty Quantification of Thermal-Optical Organic/Elemental Carbon Measurements

Analyse des points fractionnés et quantification de l'incertitude des mesures du carbone organique/élémentaire thermique-optique

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon ...

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Traitement de l'embryon, de la coquille d'œuf et de la culture fongique pour la microscopie électronique à balayage

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...